The development of Sanaria's aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoite (SPZ)-based products has received international recognition during the past year, due principally to four findings: 1) PfSPZ Challenge (infectious PfSPZ) induced malaria in volunteers, 2) PfSPZ Vaccine (radiation attenuated PfSPZ) induced unprecedented levels of PfSPZ-specific, IFN-?-producing CD8+ T cells in livers of non-human primates (NHPs), 3) purified, cryopreserved P. yoelii (Py) SPZ induced 71-100% protection in mice, and 4) our colleagues in the Netherlands' report that PfSPZ infection of volunteers taking chloroquine induced 100% protection that lasted for at least 28 months. Trials of PfSPZ Vaccine administered intravenously (IV) are now planned for the NIAID, NIH Vaccine Research Center, Bethesda, USA, and the Ifakara Health Institute (IHI) in Tanzania, and PfSPZ Challenge trials are planned for Tanzania, UK, USA, the Netherlands, Germany, and Switzerland, and PfSPZ Cvac (PfSPZ Challenge administered with chloroquine) in the Netherlands and USA. PySPZ studies in mice and data from in vitro assays with PfSPZ and PySPZ indicate potency of cryopreserved SPZ is 2.7 to 6-fold less than that of fresh SPZ. All of Sanaria's products rely on stabilization and cryopreservation of aseptic, purified PfSPZ. Improvements to stabilization/cryopreservation are critically important and when implemented will double the infectivity and potency of PfSPZ, reducing the cost of manufacturing and quality control release assays for lots of PfSPZ products by increasing the size of lots, and reducing vaccine loss/wastage in the clinic by prolonging the time after thawing that a vial of PfSPZ product can be used. Our goal is to reduce the cost of goods (COGS) overall by at least 75%. Five Specific Aims encompass work to accomplish this goal: Aim 1. Reduce by 50% the numbers of PySPZ required to achieve 100% infection and 80% protective efficacy in vivo: In vitro assays and the in vivo infectivity (ID50) and protection against challenge are the readouts. Aim 2. Increase by 50% the infectivity and potency of cryopreserved PfSPZ: Methods developed Aim 1 will be applied to PfSPZ using a combination of in vitro assays for readouts. Aim 3. Establish in NHPs that PfSPZ, cryopreserved by methods optimized in Aims 1 and 2 as compared to PfSPZ manufactured using our current methods induce a significant increase in the frequency of INF-?-secreting CD8+ T cells when used to stimulate PBMCs from immunized NHPs in vitro and when used to immunize NHPs in vivo. Aim 4. Develop the capacity to create batches of in process PfSPZ material at multiple, defined intervals that can be combined into PfSPZ bulk product and then large lots of PfSPZ final product. The goal is to extend the process hold-time up to 6 days and to increase lot size to 180,000 doses. Aim 5. Extend PfSPZ survival in the clinic post-thaw and formulation: Cryopreserved, thawed PySPZ and PfSPZ in diluent formulations designed to extend in-clinic stability will be tested using in vivo and in vitro assay with the goal of increasing to a minimum of four hours, stability in the clinic.